The field of this invention is molecular biology, particularly in the area of retrotransposons, nucleotide sequence encoding integrase therefrom, and molecular genetic methods based thereon.
Mobile genetic elements that replicate by reverse transcription are ubiquitous among eukaryotic genomes. These elements, collectively called retroelements, include the retroviruses and two classes of retrotransposons, which are distinguished by whether or not they are flanked by long terminal direct repeats (LTRs) (Xiong and Eickbush 1990). A common step in retroelement replication involves the integration of an element cDNA into the host genome (Brown and Varmus 1989). For the retroviruses and LTR retrotransposons, this step is carried out by a nucleoprotein complex called the integration complex. A key component of the integration complex is the element-encoded integrase protein, which carries out the cutting and joining steps of the integration reaction. Although, in general, no specific sequences are required at the target site for integration, the distribution of LTR retroelements is clearly non-random (Craigie 1992; Sandmeyer et al. 1990).
Examples of retroelement target specificity are provided by the retrotransposons of Saccharomyces cerevisiae. S. cerevisiae has five distinct retrotransposon families, designated Ty1-Ty5, which vary extensively in copy number (from 25-30 Ty1/Ty2 insertions to zero to two Ty5 insertions per haploid genome) (Boeke and Sandmeyer 1991; Zou et al. 1995). A distinctly non-random distribution of Ty1-Ty4 insertions has been revealed from the nucleotide sequences of several S. cerevisiae chromosomes (e.g. chromosome (chr) III (Ji et al. 1993; Oliver et al. 1992)). Most native Ty1-Ty4 elements are found within 1 kb upstream of genes transcribed by RNA polymerase III (pol III), such as tRNA genes. For Ty1 and Ty3, this genomic organization is the consequence of targeted integration; pol III genes are the preferred targets of de novo Ty1 and Ty3 transposition events Chalker and Sandmeyer 1990; Chalker and Sandmeyer 1992; Ji et al. 1993; Devine and Boeke 1996).
Mechanisms that dictate Ty target specificity have been studied in detail for Ty3. Ty3 integration is highly precise, and typically occurs within the first few base-pairs of the start site of pol III gene transcription (Chalker and Sandmeyer 1992). For tRNA genes, mutations in the promoter that abolish transcription also abolish targeted transposition. Biochemical analyses, including in vitro transposition assays, have demonstrated that the pol III transcription factors TFIIIB and TFIIIC are sufficient for targeted integration (Kirchner et al. 1995). Current models suggest that these transcription factors tether the Ty3 integration complex to its target through protein--protein interactions.
Integration sites for several retroviruses tend to be associated with DNase I hypersensitive sites, suggesting retroviruses prefer open chromatin (Craigie 1992; Sandmeyer et al. 1990). The yeast two-hybrid system has recently been used to identify a human protein, Ini1, that specifically interacts with HIV integrase (Kalpana et al. 1994). Ini1 is a homologue of the yeast transcription factor SNF5, which is known to remodel chromatin in yeast to promote transcription. The HIV integrase/Ini1 interaction suggests that retroelements may, in general, recognize specific DNA-bound protein complexes to choose their integration sites.
In striking contrast to the Ty1-Ty4 families, endogenous Ty5 insertions are located in sub-telomeric regions or on chr III adjacent to the silent mating locus HMR (Zou et al. 1995). However, none of the Ty5-related insertions in the S. cerevisiae genome characterized to date are full-length, functional transposable elements. Most characterized Ty5-related insertions in S. paradoxus are incomplete and nonfunctional as well.
The telomeres and the transcriptional silencers flanking HMR direct the assembly of a distinct type of chromatin (silent chromatin) that represses the transcription of adjacent genes (Laurenson and Rine 1992). Silent chromatin is also assembled at HML, a second mating locus on chr III, and transcriptional repression of both HML and HMR prevents the expression of mating type information unless it is copied to MAT, a third, transcriptionally active mating locus. A number of proteins contribute to silent chromatin structure and transcriptional repression at the telomeres and silent mating loci. Among these are proteins involved in DNA replication (the origin recognition complex (ORC); (Bell et al. 1993; Foss et al. 1993; Micklem et al. 1993)), transcription factors (RAPPolII1 and ABF1; (Diffley and Stillman 1988; Kurtz and Shore 1991), histones (H3 and H4; (Kayne et al. 1988; Thompson et al. 1994)), components of acetyltransferases (NAT1 and ARD1; (Mullen et al. 1989) and several proteins that have a specific role in silencing (SIR1-SIR4 and RIF1; Rine and Herskowitz 1987; Hardy et al. 1992)). Chromatin at the telomeres and silent mating loci is analogous to heterochromatin in other eukaryotes (Hecht et al. 1995). In Drosophila melanogaster, a number of transposable elements are associated with heterochromatin (Pimpinelli et al. 1995), including DNA transposons such as the P elements, which preferentially transpose to some heterochromatic sites (Karpen and Spradling 1992). Surprisingly, some heterochromatic transposable elements have evolved apparently essential roles for the cell; the HeT and TART retroelements can serve as D. melanogaster telomeres (Biessmann et al. 1990; Levis et al. 1993).
The present disclosure provides the isolation of a transpositionally active Ty5 element from the yeast, Saccharomyces paradoxus and a Ty5 transposition assay which can be carried out in S. cerevisiae, as well as other applications based on Ty5 and derivatives thereof.